Chapter 13 Recombinant DNA: Cloning and Creation of Chimeric Genes to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

Outline 13.1 Cloning 13.2 DNA Libraries 13.3 Polymerase Chain Reaction (PCR) 13.4 Recombinant DNA Technology

13.1 Cloning Clone: a collection of molecules or cells, all identical to an original molecule or cell To "clone a gene" is to make many copies of it - for example, in a population of bacteria Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible

Naturally occurring extrachromosomal DNA Plasmids Naturally occurring extrachromosomal DNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction enzymes, leaving sticky ends Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

Plasmids that can be modified to carry new genes Cloning Vectors Plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have a replicator (origin of replication) a selectable marker (antibiotic resistance gene) a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

Named for mythological beasts with body parts from several creatures Chimeric Plasmids Named for mythological beasts with body parts from several creatures After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted Ends of the plasmid/fragment are closed to form a "recombinant plasmid" Plasmid can replicate when placed in a suitable bacterial host See Figure 13.3

Often one desires to insert foreign DNA in a particular orientation Directional Cloning Often one desires to insert foreign DNA in a particular orientation This can be done by making two cleavages with two different restriction enzymes Construct foreign DNA with same two restriction enzymes Foreign DNA can only be inserted in one direction See Figure 13.6

13.2 DNA Libraries Sets of cloned DNA fragments that together represent the genes of a particular organism Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell Can't isolate it directly Trick is to find the fragment or fragments in the library that contain the desired gene

The probabilities are staggering! DNA Libraries - II The probabilities are staggering! Consider the formula on page 406 for probability of finding a particular fragment in N clones Suppose you seek a 99% probability of finding a given fragment in N clones of 10 kbp fragments If your library is from the human genome, you would need 1,400,000 clones to reach 99% probability of finding the fragment of interest!

Colony Hybridization A way to screen plasmid-based genome libraries for a DNA fragment of interest Host bacteria containing a plasmid-based library of DNA fragments are plated on a petri dish and allowed to grow overnight to form colonies Replica of dish made with a nitrocellulose disk

Colony Hybridization Disk is treated with base or heated to convert dsDNA to ssDNA and incubated with probes Colonies that bind probe (with P-32) hold the fragment of interest

Another way to find desired fragments Southern Blots Another way to find desired fragments Subject the DNA library to agarose gel electrophoresis Soak gel in NaOH to convert dsDNA to ssDNA Neutralize and blot gel with nitrocellulose sheet Nitrocellulose immobilizes ssDNA Incubate sheet with labelled oligonucleotide probes Autoradiography should show location of desired fragment(s)

The Polymerase Chain Reaction What if you don't have enough DNA for colony hybridization or Southern blots? The small sample of DNA serves as template for DNA polymerase Make complementary primers Add primers in more than 1000-fold excess Heat to make ssDNA, then cool Run DNA polymerase (usually Taq) Repeat heating, cooling, polymerase cycle